Two-chamber diffusion cell

Several investigators have used the two-chamber diffusion cell configuration. This experimental method has been found useful to determine diffusion coefficients [11] and to study drug transport from drug delivery devices [12],... 

Srinivasan et al. [17] have described a four-electrode potentiostat system which is suited to maintaining a constant voltage drop across a membrane in a two-chamber diffusion cell. This system was evaluated in connection with trans-dermal iontophoretic drug delivery of polypeptides. 

H. L. G. M. Tiemessen, H. E. Bodde, M. Van Koppen, W. C. Bauer, and H. E. Junginger, A two-chambered diffusion cell with improved flow through characteristics through for studying the drug permeation of biological membranes, Acta Pharm. Techn. 34 99-101 (1988). 

Three sets of two-chamber diffusion cell experiments were conducted 1) ethanol/saline in the donor chamber and saline in the receiver chamber, 2) saline in the donor and ethanol/saline in the receiver chamber, and 3) with ethanol/saline in both chambers. The results were shown to deviate enormously from the classical lipid barrier model. A new model, based on diffusion across a binary solvent mixture, was used to analyze the data. A good agreement was observed between experimental data and theoretical results. 

Diffusion Cells. The two chamber diffusion cells (9) were assembled by a No. 18 spring clamp with the hairless mouse3skin sandwiched in between. The volume of each half cell was 2.0 cm. An 8 mm stirrer made of stainless steel and equipped with a small teflon propeller was driven by a 150 rpm constant speed motor (Hurst, Princeton, IN) was utilized for stirring. The assembled cell was then immersed in a 37°C heated water bath (Haake, Karlsruhe, W. Germany), so that the stirring and sampling ports were the only components above the water surface. The diffusion cell was kept for 10 minutes in the water bath to allow temperature equilibrium prior to any experiment. Then ethanol/saline mixtures preheated to 37°C were pipetted into the cell to start an experiment. 

Permeability Studies. A two-chamber diffusion cell procedure (8. 12) was employed with freshly excised hairless mouse skin. The diffusion cell consisted of two half cells or compartments (2 ml volume/half cell) and the area for diffusion between the two half cells was approximately 0.6 cm2. The full-thickness skin membrane was prepared by sacrificing the mouse as previously described and excising two square sections of the left and right abdominal skin. A skin membrane was mounted between the two compartments of the diffusion cell with the dermis side facing the receiver compartment, clamped and excess skin trimmed. The assembled diffusion cells were immersed in a water bath at the selected temperature between 10 and 60°C. Glycerine baths were used for temperatures between 60 and 90°C. Each compartment was filled with PBS adjusted to 7.3 pH at the specific experimental temperatures. 

Several investigators have used a two-chamber diffusion cell apparatus to characterize in vitro scleral permeability (3-5,22-25) to radioactively—or fluores-cently—labeled compounds. 

The primary approach to assess dermal absorption is the in vitro diffusion cell. In thi,s model, skin sections (full thickness, deimatomed to a specific thickness) are placed in a two-chambered diffusion cell in which receptor fluid is placed in a reservoir (static cells) or perfused through a receiving chamber (flow-through cells) to simulate cutaneous blood flow. Chemical may either be dosed under ambient conditions neat or dissolved in a vehicle (Franz and Bronaugh cells) or in water (sLdc-by-side diffusion... 

A two-chamber diffusion cell has fluid on both sides of the skin and is used to apply a large (infinite) dose of test compound to one side of skin that will eventually result in a maximum, steady-state rate of absorption through skin. This information can be useful when infinite doses are applied to skin, such as in a transdermal delivery device. 

To measure gas and water vapor permeability, a film sample is mounted between two chambers of a permeability cell. One chamber holds the gas or vapor to be used as the permeant. The permeant then diffuses through the film into a second chamber, where a detection method such as infrared spectroscopy, a manometric, gravimetric, or coulometric method isotopic counting or gas-liquid chromatography provides a quantitative measurement (2). Die measurement depends on the specific permeant and the sensitivity required. 

Figure 5 The Costar Transwell system with a cell monolayer grown on a porous polycarbonate filter that is mounted onto a removable plastic insert forming the apical chamber. Two other systems, (1) the Costar diffusion chamber system, where a filter-grown (Snap-well) cell monolayer is sandwiched between two chambers of equal volume and the bathing solutions are agitated and/or gassed and (2) filter-grown cell monolayers mounted in a two-chamber rotating cylinder device (Imanidis et al., 1996), are not shown.

While Franz-type diffusion cells are commonly used to assess in vitro penetration of compounds across the skin, they have also been used for the assessment of compound permeability across the buccal mucosa [19, 71, 104], In this system, buccal mucosa is sandwiched between two chambers, and compound solution is added to the donor chamber with compound-free buffer in the receptor chamber. The receptor chamber is then periodically sampled to assess the amount of compound that has permeated the tissue over time. 

OECD has adopted an in vitro test for skin absorption potential (OECD TG 428, Skin Absorption In Vitro Method). According to this guideline, excised skin from human or animal sources can be used. The skin is positioned in a diffusion cell consisting of a donor chamber and a receptor chamber, between the two chambers. The test substance, which may be radio-labeled, is applied to the surface of the skin sample. The chemical remains on the skin for a specified time under specified conditions, before removal by an appropriate cleansing procedure. The fluid in the receptor chamber is sampled at time points throughout the experiment and analyzed for the test chemical and/or metabolites. 

PG release from the two delivery systems was monitored using an automated, in vitro diffusion cell system. The devices were clamped in vertical glass diffusion cells with the releasing surface of the system facing into the receptor chamber. The latter contained NaCl-Na citrate buffer and was continuously perfused at 10 ml/hr. Perfusate was collected hourly for up to 48 hours. The receptor phase of the diffusion cell was magnetically stirred and was kept at 37°C. Samples were analyzed by liquid scintillation counting. For each delivery system, 6 replicates were run and the data provided both cumulative PG released and PG release rate per unit time. 

Example 10.5 Diffusion cell and transference numbers The diffusion cell shown in Figure 10.2 has NaCl mixtures in the two chambers with concentrations c1A = lOOmmol/L and c1B = lOmmol/L. The mobilities of Na+ and Cl- ions are different and their ratio yields their transference numbers b+lb = t+/t = 0.39/0.61 (NaCl). The transference number t for an ion is the fraction of the total electric current carried by the ion when the mixture is subjected to an electric potential gradient. For monovalent ions, we have t+lt = 1. Estimate the diffusion potential of the cell at steady-state conditions at 298 K. Assume that activity coefficients are equal in the two reservoirs (Garby and Larsen, 1995). 

Example 10.6 Estimation of flow in a diffusion cell Each chamber of the diffusion cell shown in Figure 10.2 has an aqueous solution of NaCl with concentrations c1A = cXB = 100 mmol/L at 298 K. An electric potential difference of 100 mV is established between the two chambers. Estimate the diffusion flow of NaCl and its direction if... 

Before launching wound study, the ability of ATP-vesicles to penetrate the tissue was tested in skin penetration. The rat skin, which is known to have similar permeability characteristics as those of humans (19), was mounted in the FDC-6 Franz Diffusion Cells (26-28). ATP-vesicles or free ATP solution was placed in the donor dome and the receiving chamber was filled with neutral buffer. ATP, ADP, AMP and their metabolites were measured by HPLC using a modified technique described previously (29-31) in the two chambers at 2, 4, and 24 h and the contents were compared to obtain the permeability ratio. The result indicated that the ATP-vesicles dramatically increased nucleotide penetration through the skin (dermis and epidermis) by 10-20-fold (N=9, Fig. 5). 

In a diaphragm cell diffusion occurs through a horizontal porous diaphragm separating two chambers containing liquid mixtures of different compositions. In the mathematical analysis of diffusion cell data, the following assumptions normally are made. 

With the difficulties associated with accurate estimation of permeability based only on physicochemical properties, a variety of methods of measuring permeability have been developed and used, among which are (l)cul-tured monolayer cell systems, such as Caco-2 or MDCK ( 2 diffusion cell systems that use small sections of intestinal mucosa between two chambers (3) in situ intestinal perfusion experiments performed in anesthetized animals such as rats and (4)intestinal perfusion studies performed in humans (40,54-62). All of these methods offer opportunities to study transport of drug across biological membranes under well-controlledconditions. Caco-2 mono-layer systems in particular have become increasingly commonly used in recent years and human intestinal perfusion methods are also becoming more commonly available. Correlations between Caco-2 permeability and absorption in humans have been developed in several laboratories (63-72). As shown in Fig. 

Methods. The diffusion experiments were performed at room temperature (23 C) utilizing a glass diffusion cell consisting of two compartments each with a volume of 175 ml. Each chamber was stirred at a constant rate to reduce boundary layer effects. Solute concentrations were monitored by h or C tracers, refractive index, or U.V. spectroscopy. Partition coefficients, defined as the ratio of the concentrations in the membrane and in the bulk aqueous phase were determined by solution depletion technique. 

Filter assays have been very popular for identifying soluble compounds that stimulate locomotion (reviewed in [424, 426]). Two chambers are separated by a filter with pore sizes less than the diameter of the cells, such that cells must actively migrate to get through. The apparatus is oriented such that cells are put in the top chamber and can settle onto the filter. Solution with chemoattractant is put in the bottom chamber, and as it diffuses through the pores a concentration gradient is established such that the cells crawl through the filter. There are two main types of filter... 

Another very common techniqne is based on the use of an infrared sensor as described by ASTM F1249-90 [23]. The barrier sample is sealed between a dry and a wet chamber kept at known temperature and relative humidity. The two chambers make up a diffusion cell which is placed in a test station where the dry chamber and the top of the barrier are flushed with dry air. 

In the left side, gas A flows in the direction as shown and picks up B due to the diffusion of B from the other side of the porous medium. Similarly, B at the other side will pick up A from its diffusion through the porous medium. The flow rates of the two sides can be carefully adjusted to give zero pressure gradient across the media (that is the total pressure is uniform throughout the porous medium). The concentrations of gases A and B are analysed by detectors, such as the thermal conductivity cell, and then the diffusive fluxes of A and B can be calculated. This is the steady state method. Recently, this method was extended to allow for transient operation such as a step change or square pulse in one chamber and the response is monitored in the other chamber. With this transient operation, the contribution from the dead end pore can be studied. This contribution is not seen by the steady state method, but its advantage is the ease of operation under isothermal operations. Detailed analysis of diffusion cell under steady state and transient conditions is provided in Chapter 13. 

The last chapter shows the utility of the time lag method and its applications in the characterization of diffusion and adsorption of pure component systems. In this chapter we address the diffusion cell method, which is used mainly with systems containing two solutes. The process involves the counter-diffusion of these two solutes through a porous medium from one chamber to the other. Usually both of these chambers are open, but there are applications where one of the chambers is a closed chamber. There are two modes of operation of the diffusion cell method. One is the steady state diffusion cell, and the other is the transient diffusion cell. 

The steady state diffusion cell was first developed by Wicke and Kallanbach in 1941. Hereafter we shall refer this method as the WK method. In their method, a pellet or many pellets are mounted in parallel between two open chambers (Figure 13.1-1). In one chamber, one component (labelled as A) is flowing into and out of the chamber by convection, and in the other chamber another component (B) is also flowing into and out of that chamber. The residence time in these two chambers are usually much smaller than the diffusion time through the pellet. The species A diffuses through the pellet in the opposite direction to the diffusion path of the species B. The pressures of the two chambers are maintained the same, and hence the counter-flows of the two species are by the mechanism of combined molecular diffusion and Knudsen diffusion. The fluxes of these solutes are calculated by simply measuring the concentrations of A and B in the exit streams of the two chambers. This steady state method of Wicke-Kallanbach is very simple to carry out and it provides a simple means to calculate steady state fluxes through the pellet. However, it does have a number of problems ... 

The steady state diffusion cell is composed of a porous medium bound by two chambers. The porous medium can be either a particle or a collection of particles mounted in parallel as shown in Figure 13.1-1. The advantages and disadvantages of the steady state diffusion cell are listed in the following table. 

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